Hara et al. BMC Biology (2018) 16:40 https://doi.org/10.1186/s12915-018-0509-4 RESEARCH ARTICLE Open Access Madagascar ground gecko genome analysis characterizes asymmetric fates of duplicated genes Yuichiro Hara1,2, Miki Takeuchi3†, Yuka Kageyama1,4†, Kaori Tatsumi1,2, Masahiko Hibi3,5, Hiroshi Kiyonari6,7,8,9 and Shigehiro Kuraku1,2* Abstract Background: Conventionally, comparison among amniotes – birds, mammals, and reptiles – has often been approached through analyses of mammals and, for comparison, birds. However, birds are morphologically and physiologically derived and, moreover, some parts of their genomes are recognized as difficult to sequence and/ or assemble and are thus missing in genome assemblies. Therefore, sequencing the genomes of reptiles would aid comparative studies on amniotes by providing more comprehensive coverage to help understand the molecular mechanisms underpinning evolutionary changes. Results: Herein, we present the whole genome sequences of the Madagascar ground gecko (Paroedura picta), a promising study system especially in developmental biology, and used it to identify changes in gene repertoire across amniotes. The genome-wide analysis of the Madagascar ground gecko allowed us to reconstruct a comprehensive set of gene phylogenies comprising 13,043 ortholog groups from diverse amniotes. Our study revealed 469 genes retained by some reptiles but absent from available genome-wide sequence data of both mammals and birds. Importantly, these genes, herein collectively designated as ‘elusive’ genes, exhibited high nucleotide substitution rates and uneven intra-genomic distribution. Furthermore, the genomic regions flanking these elusive genes exhibited distinct characteristics that tended to be associated with increased gene density, repeat element density, and GC content. Conclusion: This highly continuous and nearly complete genome assembly of the Madagascar ground gecko will facilitate the use of this species as an experimental animal in diverse fields of biology. Gene repertoire comparisons across amniotes further demonstrated that the fate of a duplicated gene can be affected by the intrinsic properties of its genomic location, which can persist for hundreds of millions of years. Keywords: gecko, phylome, gene repertoire evolution, gene loss, gene duplication, disparity of genomic fields Background birds, which are descendants of a reptilian lineage that Reptiles and birds, together composing the evolutionary originated from dinosaurs, have a variety of highly derived clade Sauropsida, are the evolutionarily closest living com- morphological characters, including feathers, wings, and panions to mammals [1]. In modern biology, chicken has toothless beaks. Furthermore, recent studies revealed that been established as the primary study system to address regional characteristics in avian genomes could hinder se- the origin of characters shaping mammals [2]. However, quence assembly in those or some regions, resulting in the exclusion of hundreds of genes [3–5] that were previously considered to be evolutionarily lost [6]. Therefore, in * Correspondence: [email protected] †Equal contributors order to conduct comparative analyses, the use of reptiles 1Phyloinformatics Unit, RIKEN Center for Life Science Technologies, Kobe, in place of birds as the closest relatives to mammals is a Hyogo 650-0047, Japan reasonable choice in phylogenetic contexts. Nevertheless, 2Laboratory for Phyloinformatics, RIKEN Center for Biosystems Dynamics Research, Kobe, Hyogo 650-0047, Japan reptiles currently do not serve as practical study systems Full list of author information is available at the end of the article © Kuraku et al. 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Hara et al. BMC Biology (2018) 16:40 Page 2 of 19 with regards to feasibility in operational experiments and consolidated the orthology/paralogy inference of human accessibility to comprehensive molecular sequence infor- Nodal [12]andOct4 [13] genes to the corresponding mation. The Madagascar ground gecko (Paroedura picta; chicken genes. A comprehensive set of gene phylogenies is Fig. 1a), belonging to the family Gekkonidae, is a reptile referred to as a phylome [14], and reconstructing a phy- candidate that meets these requirements. In captivity, lome with a broad taxon sampling is applicable to an in- females of this species lay two eggs every 10 days all year tensive search for genes whose orthologs are absent from round [7], which allows a stable supply of embryonic ma- model mammalian and bird genomes but are present in terial. Additionally, the relatively thin eggshells enable in some reptiles; indeed, these genes could be thought to ovo manipulation [7, 8], and embryonic stages of this have been lost in the common ancestor of amniotes if rep- species have already been categorized [7]. With these de- tilian genomes are not employed in the search. In this velopmental biology advantages, embryos of this gecko strategy, genes missing in genomic sequences of multiple species are becoming frequently used for various studies lineages can be systematically analyzed, providing an in this field, revealing the evolutionary histories of insight into the mechanisms of gene repertoire diversifica- molecular mechanisms of cortical neurogenesis and gas- tion. However, studies of the evolution of gene repertoire trulation in amniotes [9, 10], which has not yet been have thus far largely concentrated on gene duplication. accomplished using only mammals and birds. Among rep- In this study, we conducted de novo whole genome se- tiles, the green anole is relatively popular in biological re- quencing of the Madagascar ground gecko. By including search, especially at the molecular level, supported by its the inferred genes in its genome, we have reconstructed genomic sequence resources. However, its seasonal breed- a phylome. Remarkably, our results uncovered hundreds ing and soft eggshell [11] have limited the conduct of of genes retained by reptiles but independently absent continuous and operational experiments. Given these from both mammalian and avian genomes. Comparing issues, genome sequences of the Madagascar ground these genes with their paralogs, we discussed causal fac- gecko, with higher feasibility in experimentation, will tors of the asymmetric fates of these paralogs during facilitate molecular comparative studies involving this vertebrate evolution. species in diverse research fields. In addition to the comparison of morphologies, a rea- Results sonable taxon sampling in phylogenetic contexts can re- Genome sequencing vise evolutionary scenarios of gene repertoire in amniotes The P. picta genome was sequenced with 75× coverage as previously proposed through the use of fewer species. For paired-end and mate-pair libraries on the Illumina platform example, molecular phylogenetic trees including reptiles (Additional file 1: Table S1), resulting in a genome assembly a b Madagascar ground gecko c Fig. 1 Quality assessment of the P. picta genome assembly. (a) P. picta adult individuals with different color phases. (b) A scatter plot of the assessments of assembly quality: CEGMA completeness scores referring to the CVG and N50 scaffold lengths for available reptile genome assemblies. Each reptile genome assembly is shown with a dot. Detailed statistics of assembly quality are included in Additional file 1: Table S2. These metrics were calculated with the webserver gVolante [90]. (b) Synteny conservation between the longest scaffold of the P. picta genome assembly (scaffold00000001; 33 Mb long) and a part of the green anole chromosome 3 (10–48 Mb in 204 Mb long). Green and gray boxes are protein-coding genes in the gecko and anole genome assembly, respectively, and blue bands indicate orthology between gecko and anole genes Hara et al. BMC Biology (2018) 16:40 Page 3 of 19 of 1.69 Gb (Table 1). This size was close to the measure of harbors unusually low intragenomic GC heterogeneity its haploid nuclear DNA content in somatic cells of P. picta [18]. The overall GC content of the P. picta genome was (1.80 Gb; Additional file 1:TableS3).Thegenomeassembly higher than that of the green anole genome and compar- had an N50 scaffold length of 4.1 Mb and an overall GC able to those of the turtle and crocodile genomes (Add- content of 44.8% (Table 1). A total of 37.3% of the genome itional file 3: Figure S2a). However, the variance in GC assembly consisted of repetitive elements (Additional file 1: content distribution in the P. picta genome was close to Table S4, Additional file 2:FigureS1).Genepredictionwas that of the green anole genome, especially when using performed with the program Augustus, incorporating the narrow sliding windows (Additional file 3: Figure S2b). optimized prediction parameters for the species and predic- A principal component analysis of diverse amniote ge- tion hints based on transcript evidence and protein-level nomes on the frequencies of 3-mer nucleotides demon- homology to other amniotes.